Ongoing microvascular injury is a hallmark of systemic sclerosis (SSc). The injury is associated with microthrombi formation secondary to platelet aggregation. One of the initial events in hemostasis is the interaction of platelets with the underlying collagen in damaged vessels. The specific mechanism of collagen induced platelet aggregation and its role in pathological thrombosis are not well understood. Recently, nitric oxide (NO) has also been reported to be involved in the collagen-platelet interactive process. The role of NO in cellular signaling has become one of the most rapidly growing areas in biology. In many instances NO mediates its biological effects including platelets. We propose 1 ) to define the structural features of the CIR and CIIIR that are essential for binding to CI and CIII, 2) to develop synthetic oligopeptides patterned after deduced CIR and CIIIR protein amino acid sequences that inhibit binding of CI and CIII to normal platelets and determine their effect on the binding of SSc platelets to CI and CIII. The results obtained in specific aim 1) will provide clues as to what size and amino acid sequence are most effective in blocking platelet interaction with CI and CIII, 3) to determine the functional significance of the recombinant protein and its signal transduction pathway, 4) to explore whether nitrosylation of specific platelet protein(s) occurs on tyrosine residues upon collagen platelet interaction of SSc patients and whether nitrosylation contributes stimulatory or inhibitory effects on platelet function in SSc patients and 5) to extend our assessment of the content of NOS in red blood cells of SSc patients. Binding assays will be used to determine the direct interaction of both [32PO4]-labeled rCIR and CIIIR and [125I]-labeled peptides to immobilized collagen on microtiter wells. Western blot with anti-nitrotyrsine will be used to detect platelet protein nitrusylation. The activity of NOS will be assayed with the conversion of [3H]-arginine to [3H]-citrulline. Functional studies will be performed by inhibiting both types I and III collagen-induced platelet aggregation and release reaction, by binding assay, protein phosphorylation, and flow experiments. We hope to understand more about the structure and functional significance of the platelet CIR and CIIIR. The defined reactive site(s) of CIR and CIIIR will aid in the design of active peptide(s), which could be used to modulate collagen-platelet interaction. These results will be important in understanding collagen-platelet interaction and may pave the way for the intervention of pathological thrombi formation in SSc.